Effect of COVID‐19 infection on sex hormone levels in hospitalized patients: A prospective longitudinal study in Iran

Abstract Introduction This study aimed to evaluate the levels of sex hormones in patients with COVID‐19 in Ahvaz, Iran. Methods A prospective longitudinal study was conducted at Razi hospital, Ahvaz, Iran, from July 2020 to Febuary 2021. The levels of sex hormones including estradiol, progesterone, luteinizing hormone (LH), follicle stimulating hormone (FSH), and total and free testosterone were measured in 162 patients with COVID‐19 infection during hospitalization and 1 month after discharge. A demographic questionnaire and a checklist were used to collect the data. Mann−Whitney U test, χ 2 test, Fisher's exact test, Wilcoxon test, and logistic regression were used to analyze the data. Results Sex hormones were assessed in 162 patients at baseline; however, a month after discharge, only 69 patients provided consent for assessment, and 9 had passed away. The estradiol level was 407.70 ± 623.37 and 213.78 ± 407.17 pg/ml in female patients with severe and moderate diseases at baseline, respectively which reduced to 195.33 ± 380.04 and 58.20 ± 39.45 pg/ml after discharge (p = 0.011 and p = 0.001). The alteration in the levels of progesterone, LH, and FSH were not significant. The level of LH in both groups of male patients with severe (6.64 ± 2.91 IU) and moderate disease (6.42 ± 4.44 IU) was high, which reduced after discharge (4.16 ± 2.44 and 3.93 ± 3.15 IU, respectively), but this decrease was significant only in the patients with severe disease (p < 0.0001). The alteration of FSH and free testosterone were not significant. The level of testosterone was 1.19 ± 0.73 and 1.46 ± 1.22 ng/ml at baseline in patients with severe and moderate diseases which increased to 2.64 ± 1.25 ng/ml, p < 0.0001, and 2.54 ± 0.93 ng/ml, p = 0.001, respectively after discharge. Conclusion Our findings showed that the level of estradiol in female patients increased significantly while the level of testosterone in male patients decreased during the active phase of infection. Due to the attrition of patients in the follow‐up period, more studies are needed to confirm these results.

There is limited evidence that women are less likely to contract COVID-19 infection, and in case of infection, they show less severe disease symptoms such as thromboembolic events compared with men. 5,6 Data from some countries such as Italy which has experienced high rates of COVID-19 infection have also shown that the mortality of men is more than that of women in all ages. 7 The hypothalamus-pituitary-adrenal axis has been reported as the main target of COVID-19 infection, and glucocorticoid has been suggested by almost all treatment regimens for the patients with moderate or severe forms of COVID-19 infection. 8 Since angiotensin-converting enzyme-2 (ACE2) receptor is expressed in endocrine organs and is also used by SARS-CoV-2 to attack the host cells, there is a hypothesis that endocrine organs and their hormones may be affected by this virus. 9 The relationship between COVID-19 infection and sex hormones has shown that female hormones such as estrogen and progesterone play a protective role in COVID-19 infection in women. Mauvais-Jarvis et al., 10 for instance, found that steroids 17β-estradiol (E2) and progesterone have a protective role against COVID-19 infection in women. In addition, Li et al. 11 reported that sex hormone concentration and anti-mullerian hormone in childbearing women did not change with COVID-19 infection. However, other studies have shown decreased levels of male hormones in patients with COVID-19 infection. Dhindsa et al. 12 for example, studied 152 hospitalized patients and found that the patients had significantly low levels of testosterone. Also, the serum level of total testosterone in male patients with severe disease and mostly in those admitted to the intensive care unit (ICU) was significantly lower than that in patients with a mild infection. 13 Given the limited number of studies on the relationship between sex hormones and COVID- 19

| Setting
All patients were recruited from Razi Hospital in Ahvaz, Iran. Razi Hospital is a university hospital with 200 beds that was designated to admission of patients with COVID-19 infection from the beginning of the pandemic. Ahvaz is the capital of Khuzestan province in Iran and has a population of 1,244,000 according to the latest official census. 14 Data collection was started in July 2020 and completed in February 2021 that was coincident with the second and third surges of COVID-19 in Iran. 15

| Sample size
The necessary sample size for female participants was calculated based on a pilot study. Minimum sample size based on alteration of progesterone levels in infected women with COVID-19 was calculated from the following formula: A severe disease was defined when the patient had a lung infiltration >50%, or if the level of blood oxygen was less than 93%, and a respiratory rate was more than 30 breaths/min. Severity of disease was confirmed by chest CT-scan. All patients with these criteria were transferred to the ICU. The severity of disease was confirmed by an infectious disease specialist.

| Outcomes
The changes in sex hormones in male and female hospitalized patients were outcomes of this study.

| Study variables
COVID-19 infection was the independent variable and female and male sexual hormones were dependent variables.

| Procedure
Five milliliter venous blood were drawn from hospitalized patients and placed into gel tubes, and transferred to a reference laboratory (Novin Lab). Then the blood samples were centrifuged for 10 min and the sera were separated in gamma tubes with a lead and stored

| RESULTS
In the initial assessment, 162 patients were assessed in terms of their serum levels of sex hormones. One month after discharge, 9 patients passed away (the mortality rate was 5.5%), and only 69 patients provided consent for vein puncture.  High blood pressure 8 (4.9) 6 (4.5) 2 (6.7)

| Alteration of sex hormones in female patients
Heart disease 6 (3.7) 6 (4.5) 0 (0)  The relationship between severity of disease and the level of hormones was assessed using logistic regression, and the results are presented in Table 3. As evident from this table, there was no significant relationship between level of hormones and the severity of the disease. Although the levels of testosterone and free testosterone were lower in male patients with chronic disease at baseline and 1 month after discharge, the differences were not significant.

| Alteration of sex hormones in male patients
No correlation was observed between duration of the disease and hormone levels (

| DISCUSSION
This study aimed to compare the level of sex hormones in hospitalized male and female patients with COVID-19 upon hospitalization and 1 month after discharge. Our results indicated that the level of estradiol was significantly high in female patients with severe or moderate diseases during hospitalization, but it reduced after discharge. The level of progesterone was high during hospitalization among patients with severe disease but reduced after discharge, while in patients with moderate disease this alteration was negligible. Although the levels of LH and FSH increased at baseline and decreased after discharge, these alterations were not significant. Estrogen is well-known to have a protective role in the women's body functions, especially against viral infections, and it has also been reported that this hormone can activate humoral immunity. 16   conducted on a small sample of patients.

T A B L E 3 Relation between baseline hormone levels and severity of infection in hospitalized patients
In addition to the role of hypothalamus in sex hormone alteration, the impact of mental disorders such as stress and depression on sex hormones cannot be ignored. 24 In the present study, 34 out of 162 patients had severe disease and were transferred to the ICU. However, due to the small sample size and lack of sufficient studies in this area, we cannot draw a definitive conclusion about the relationship between sex hormone and severity of disease. In this study, we found that mortality rate is 5.5%, which is much higher than other reports from Iran. 25

ACKNOWLEDGMENTS
The expenses of this study were provided by Ahvaz Jundishapur University of Medical Sciences. The funder did not have any role in the design, data collection, data analysis and interpretation, and writing and submitting the manuscript to a journal. We would like to thank all patients who participated in this study. We also would like to thank Dr. Mahmoud Maniati as he edited this manuscript.

CONFLICT OF INTEREST
The authors declare no conflict of interest.

DATA AVAILABILITY STATEMENT
The data sets generated and/or analyzed during the current study are not publicly available due to restrictions (Ahvaz Jundishapur University of Medical Sciences does not permit to data publicity before publication) but are accessible through the corresponding author upon reasonable request.

ETHICS STATEMENT
This study was approved by the Ethics Committee of Ahvaz Jundishapur University of Medical Sciences, Ahvaz, Iran (Ref No: IR.AJUMS.REC.1399.541). All methods were performed in accordance with the relevant guidelines and regulations. All participants provided written informed consent before data collection.

TRANSPARENCY STATEMENT
The lead author Parvin Abedi affirms that this manuscript is an honest, accurate, and transparent account of the study being reported; that no important aspects of the study have been omitted; and that any discrepancies from the study as planned (and, if relevant, registered) have been explained.